Scanning Ion Conductance Microscopy: A new approach to understanding cancer cell stiffness

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The UK incidence of melanoma (skin cancer) has increased by 45% in the past decade. Melanoma is the 5th most common cancer in the UK with around 16,000 new melanoma skin cancer cases each year. However, mortality rates of melanoma in the UK have increased by 156% since the early 1970s. While an increase in incidence may be due to the more proficient clinical screening techniques, there has been little improvement in the understanding of melanoma development, prognosis and treatments. Pigmentation within the skin strongly protects against ultraviolet (UV) light-induced DNA damage. In human skin, melanin is produced by melanocytes which are present in the basal epidermis and hair follicles. Melanin within the cell acts as an UV light filter and form barriers to prevent UV light causing damage to DNA. Alterations and mutations within genes in melanocyte development and important metabolic pathways can alter the cells growth and proliferation leading to cancer formation. Melanocyte transformation into cancer and melanoma cell transformation into aggressive metastatic melanoma is associated with alterations in the mechanical properties of the cell. It is hypothesised that metastatic cells are softer than their non metastatic counterpart to allow the cell to travel in the bloodstream to secondary sites in the body and survive there. However, in the case for melanoma, this relationship is not clear as there have been studies which show that highly metastatic cell lines are stiffer than their non metastatic counterpart. In addition, a higher level of ROS has been seen in almost all cancers and is associated with tumour progression. The tumour metabolic environment influences growth and treatment response and can therefore ensure the cancer cells survive in their micro-environment and metastasise. The relationship between cancer cell stiffness and ROS is not yet known. If cell stiffness does play an important role in cancer metastasis, perhaps ROS also has a part to play in cytoskeletal alterations to allow for metastasis. Currently, the most widely used method for mapping cell stiffness is atomic force microscopy. Our group has developed a new method which is non-contact and low-force so that no damage occurs to the cell when scanning. The method is based upon scanning ion conductance microscopy (SICM) which can map cell topography with nanoscale precision. This project is the first to assess the correlation between cell stiffness and metastatic ability in melanoma cell line using established immortal melanocyte cell lines from the Functional Genomics Cell Bank at St George's. This project meets the MRC's interdisciplinary core skill as it relies upon skills in:- Biophysics - use of the SICM cell stiffness equipment (through collaboration with Imperial College London and ICAPPIC Limited)- Microscopy - SICM, fluorescence and confocal microscopy- Cell Biology - mammalian cell culture, cell proliferation assays, cell viability assays, ROS degradation assays- Molecular Biology - immunostaining, immunoblotting- Statistics - processing and analysing datasets- Presentation - presenting findings in the form of presentations, posters and peer review publicationsThis project also encompasses the MRC's quantitative core skills as there will be processing and analysis of interdisciplinary datasets. The range of known skills (molecular biology) and newly developed skills (biophysics, microscopy and cell culture) used throughout this PhD will allow the role of cell stiffness within melanoma to be understood and allow quantitative, accurate 3D mapping of cell stiffness during different stages of melanoma development and progression. The SICM system could also have a future diagnostic application in distinguishing between cancer and non-cancer cells in patients and monitor key biomarkers to optimise treatment. Keywords - Cancer, Melanoma, Cell Biology, Molecular Biology, Biomarker, Cell Stiffness, Mechanobiology